S~\1BIOSIS(2009)47,43-50
©2009 Balaban, PhiladelphialRehovot
ISSN 0334-5114
Cell proliferation and growth in Zoothamnium niveum (Oligohymenophora, Peritrichida) - thiotrophic bacteria symbiosis Ulrike Kloiber", Bettina Pflugfelder', Christian Rinke l ,2, and Monika Bright' [Department of Marine Biology, University of Vienna, Althanstrasse 14, A-1090 Vienna, Austria, Tel. +43-14277-54331, Fax. +43-14277-54339, Emails.
[email protected],
[email protected] and
[email protected]; 2Current address: School of Biological Sciences, Washington State University, Pullmann, WA 99164, USA,
[email protected]
(Received March 31, 2008; Accepted July 4, 2008)
Abstract Chemolithoautotrophic, sulphide-oxidizing (thiotrophic) symbioses represent spectacular adaptations to fluctuating environmental gradients and survival is often accomplished when growth is fuelled by sufficient nourishment through the symbionts leading to fast cell proliferation. Here we show 5'-bromo-2'deoxyuridine (BrdU) pulse labelling of vegetative growing Zoothamnium niveum, a colonial ciliate obligately associated with thiotrophic ectosymbionts, and demonstrate age related growth profiles in three heteromorphic host cell types. At the colony's apex, a large top terminal zooid performed high proliferation activity, which decreased significantly with increasing colony age but was still present in old colonies indicating that this cell possesses lifelongcell division potential. In contrast, terminal branch zooids proliferated independent of colony age but appeared to be limited by their cell division capacity predetermined by branch size, thus leading to the strict, feather-shaped colony form. Appearance of labelled terminal branch zooids allowed us to distinguish a highly proliferating apical colony region from an almost inactive, senescent basal region. In macrozooids attached to the colony, extensive BrdU labelling suggests that DNA synthesis occurs in preparation for a new generation. As motile swarmers, the macrozooids seem to be arrested in the cell cycle and mitosis and cell division occur when the swarmer settles and transforms into a top terminal zooid buildingup a new colony. Keywords:
Cell proliferation, growth, bromodeoxyuridine, ciliate, Zoothamnium, symbiosis, chemoautotrophy, thiotrophy
1. Introduction Beneficial associations between bacteria and eukaryotes exist in large numbers and varieties in marine habitats. Prominent hosts of chemolithoautotrophic, su lph ideoxidizing (thiotrophic) bacteria are invertebrates and protists occurring in sulphide rich habitats from the intertidal zone to the deep sea (Cavanaugh et al., 2006; Ott et aI., 2004). Mutual benefits driving these interactions are mainly nutritional, as the host is provided with a food source and the symbiont is able to exploit sulphide and oxygen gradients (Cavanaugh et al., 2006; Fenchel and Finlay, 1989; Fisher and Childress, 1986). In order to maintain a well functioning mutualistic association, cell division and reproduction of the host must be synchronized
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with the life cycle of the symbiont. However, our understanding of growth dynamics in thiotrophic symbioses is in its infancy. The host of a remarkable shallow-water thiotrophic ectosymbiosis is the colonial, peritrich ciliate Zoothamnium niveum (Ciliophora, Oligohymenophora) first described by Hemprich and Ehrenberg (1831) and Ehrenberg (1838) from the Red Sea. Z. niveum grows to more than one centimetre in length and is therefore, the largest known representative of a diverse genus that comprises over 70 described species (Ji et al., 2006). The sessile ciliate is obligately associated with the sulphide-oxidizing Gammaproteobacterium Candidatus Thiobios zoothamnicoli (Rinke et aI., 2006; 2007). This bacterium covers almost the entire colony's surface in a strict monolayer (Bauer-Nebelsick et al., 1996a) and gives the ciliate its conspicuous white colour. Zoothamnium niveum colonies are feather-shaped, consisting of a central stalk from which branches appear in
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an alternating arrangement, bearing three heteromorphic cell types, termed zooids (Bauer-Nebel sick et al., 1996b): (I) terminal zooids, (2) microzooids and (3) macrozooids. The colony results from a strict ramification pattern that is achieved through a specific series of longitudinal fissions: First, on the tip of each colony a top terminal zooid divides and generates a new terminal zooid which initiates the formation of a new branch. Thus, the number of branches is equivalent to the divisions of the top terminal zooid (Rinke et al., 2007) which is responsible for the length growth of the colony. Second, the new terminal zooid continues to divide and generates up to 20 structurally similar microzooids on a new segregated branch. These microzooids are left behind as feeding zooids and are equipped with a complete cytopharynx. Third, macrozooids asexually originate mitotic divisions and develop at a well determined place, generally at the bases of the branches. These voluminous zooids have the potential to transform into motile swarmers that can leave the colony and initiate a new colony after settlement (Bauer-Nebelsick et al., 1996a). Zoothamnium niveum typically inhabits mangrove peat walls along tidal channels in the Caribbean Sea. Patches of up to hundred colonies settle at fast appearing and disappearing mini-vents, where the surface of the sulphide laden peat wall is disturbed occasionally and only allows for settlement at the oxic-anoxic interface. Patch life span is limited to approximately 20 days as these spatial habitats are overgrown rapidly by a microbial community, thus closing the vents after a short time (Ott et al., 2004). Hence, this ectosymbiosis faces the challenge of habitat instability, and survival should be characterized by common life history traits, such as fast growth and early and high reproduction capabilities. In general, detailed information of life history traits in symbiotic partners is scarce due to the limited success in cultivating thiotrophic symbioses. However, the development of an artificial gradient system allowed the cultivation of the Zoothamnium niveum symbiosis for several generations (Vopel et al., 200 I). By simulating natural conditions in a flow through aquarium, experiments with these artificial sulphide-producing systems revealed a 6-8 days life span of the Caribbean host ciliate. Starting with a motile swarmer that had settled within a few hours of migration, Z. niveum developed from a single cell to a full grown colony within 4 days (Ott et aI., 2004). Furthermore, the cultivation of the symbiosis from the Mediterranean Sea in an automated flow-through respirometer during its entire lifespan allowed documentation of asexual reproduction and showed an average life span of II days when optimal sulphide conditions were provided (Rinke et al., 2007). A distinctive characteristic of colonial species within the genus Zoothamnium is the strict ramification pattern. Taking this into consideration, Z. niveum provides a particularly useful system for tracing proliferating cells. Recent studies have shown that labelling with the
thymidine analogue 5'-bromo-2'deoxyuridine (BrdU) is a suitable tool for studying proliferation kinetics in aquatic invertebrates (Alexandrova et al., 2003; Gschwentner et aL, 2001; Nakayama et al., 2005). Originally developed for cells in culture, this labelling technique has also been applied to whole-mount preparations (Plickert and Kroiher, 1988). BrdU is only incorporated into the DNA of cells that undergo DNA replication during the S-phase of the cell cycle and can be detected immunocytochemically by a specific antibody (Gratzner, 1982). Since replicative DNA synthesis is central to any increase in cell number (Moore et al., 1994), the percentage of BrdU labelled cells (proliferation index, PI) can hence serve as a dynamic measure of proliferation activity (Morris, 1993). Regarding ciliates, BrdU labelling studies have so far focused on the nuclear architecture and function of macro- and micronuclei in solitary species (Postberg et al., 2005; Tanaka and Watanabe, 2003). Investigating the host's growth profiles is one approach that can enhance our understanding of dynamic symbiotic entities such as the Zoothamnium niveum symbiosis, particularly at the cellular level. For this reason, the present study aims to identify proliferating zooids in Zoothamnium niveum by labelling whole mount colonies with BrdU. We report spatial distribution patterns of BrdU labelled zooids within the colony, and use this data to reveal their proliferation activity during the colony's life span to elucidate growth profiles in this uniquely large symbiotic protist.
2. Material and Methods
Sampling
Specimens of Zoothamnium niveum were collected in the main channel at the north end (Batfish Point) of the mangrove island Twin Cays (l6°48'N, 88°05'W; Belize Barrier Reef, Caribbean Sea) in February 2006. Colonies were cut from vertical mangrove peat walls in 0.5 to 3 m depth and transported 3.5 km seawards to the Carrie Bow Cay Marine Field Station (CCRE program of the National Museum of Natural History, Washington, DC), where labelling experiments were conducted. Proliferation labelling
For pulse labelling experiments, living specimens were treated with 5'-bromo-2'deoxyuridine (BrdU, SIGMA). Twelve specimens each were incubated for 15 min, 30 min, and 1 h in 1 mM BrdU in 0.2 urn filtered seawater. At the end of each pulse period, whole specimens were rinsed with sterile seawater (3 x 5 min), fixed with 3.2% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS, pH 7.2) overnight, dehydrated in 30, 50, and 70%
GROWTH PROFILES IN ZOO THAMNJUM NIVEUM
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Figure I. Zoothamnium niveum colony. A. General view of an old colony with more than 50 branches, showing the characteristic ramification pattern; scale bar 0.5 mm. B. Schematic drawing demonstrating the heteromorphic cell types: top terminal zooid (ttz), terminal branch zoo ids (tbz), microzooids (mic), and macrozooids (mac).
Figure 2. Light (A,C) and corresponding epifluorescence (B,D) micrographs showing the specificity of anti-BrdU antibody in macronuclei. A, B. Brdl.I-positive S-phase macronucleus (bright green label); scale bar 20 urn. C, D. Non-labelled macronucleus; scale bar 20 11m.
Figure 3. Proliferating zooids. A. General overview of colony demonstrating the characteristic, spatial distribution pattern of BrdU labelled zooids. Note that label decreases from the apical to the basal colony region; scale bar 100 urn. B. Enlargement of the top terminal zooid showing a large, labelled macronucleus; the smaller, labelled terminal branch zooid on the left side originated from the division of the top terminal zooid; scale bar 251Jm. C. Enlargement of labelled terminal branch zoo ids in the apical colony region; BrdU-positive nuclei are elongated and often show corkscrew structure; scale bar 251Jm. D. Enlargement of a labelled macrozooid. Note intense staining of the large, irregularly branched macronucleus extending through the whole cell; scale bar 25 11m.
Figure 7. Macrozooid morphology. Note the morphology of the mature macrozooid (mac; arrows indicate furrow where ciliary wreath develops) compared to the slender microzooids (mic); macrozooids in this transformation stage with developed somatic ciliature never showed BrdU label. A. Light micrograph; scale bar 50 urn, B. Corresponding epifluorescence micrograph; scale bar 50 11m.
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ethanol (each 5 min) and stored in 70% ethanol until further treatment.
Immunocytochemical detection BrdU treated specimens were rehydrated in 50 and 30% ethanol (each 10 min), transferred to PBS, permeabilized in 0.1% Triton X.IOO, PBS for 4 h at room temperature and then treated with 30 ul/ml proteinase K in PBS for 20 min at 37°C. For DNA denaturation, specimens were exposed to 0.1 M HCI (l0 min, on ice) and 2 N Hel (1 h, 37°C). After washing with PBS (3 x 5 min), unspecific staining was blocked by incubation in 3% bovine serum albumin, 0.1% Triton X·lOO, PBS (blocking solution) for 30 min at 37°e. BrdU incorporation was detected using monoclonal mouse anti-BrdU, Alexa Fluor 488 conjugate antibody (MOLECULAR PROBES) at 1:20 dilution in blocking solution overnight at 4°C. After 2 wash steps (each 10 min) in Tris high salt buffer (29.2 gIl NaCI, 4.4 g Tris/HCI, pH 7.5 and 0.5% Tween 20), whole mount specimens were mounted in PBS and sealed with nail varnish. All necessary positive and negative controls were carried out.
Analyses ofproliferating zoo ids Specimens were examined with a Nikon Eclipse E 800 epifluorescence microscope, equipped with a digital camera. Images were processed with the software program analySIS (3.2) and Adobe Photoshop (7.0). To determine a relationship between colony age and presence of proliferating zoo ids, we first counted the number of branches per colony in BrdU treated specimens (number of colonies n == 34). Branch number ranged from 6 to 128. Since length growth in Zoothamnium niveum colonies is accomplished by increasing the number of branches over time, we used the branch number to determine the approximate age of a colony and arbitrarily classified colonies with less than 50 branches as young specimens and colonies with more than 50 branches as old specimens (Fig. 1a). As only the top terminal zooid at the tip of the colony is responsible for length growth in the colony (Fig. I b), we distinguished it in the present study from the terminal zooids of the branches (terminal branch zooids). Subsequently, we discriminated an apical, a middle and a basal colony region and recorded the spatial distribution of BrdU labelled zooids within each colony (n == 34). The percentage of BrdU labelled zooids of total zooids counted is given as the proliferation index (PI) for each zooid type, respectively. In order to increase the power of statistical comparison, similar treatments were combined into a short pulse (15 and 30 min pulse, n == 24) and a long pulse (1 h pulse, n == 10) period. Since data sets did not meet the assumption of normal distribution, differences in PI were statistically analyzed using nonparametric chi-square and Mann-Whitney's U test. Multiple data sets, such as the PI
among colony regions, were compared by using the Kruskal-Wallis, followed by Tamhane's post hoc multiple comparison test for unequal variances with a 95% confidence interval (SPSS 11.5). A probability of p
